Effects of jujube (Ziziphus jujuba mill.) fruit extracts on oxidative stress: A systematic review and meta‐analysis of rodent studies

Abstract This study aimed to evaluate the effects of jujube (Ziziphus jujuba Mill.) fruit extracts on oxidative stress levels in rodent models. Animal studies meeting the inclusion criteria were retrieved from PubMed, Web of Science, Embase, China National Knowledge Infrastructure (CNKI), Wanfang Data Knowledge Service Platform, and VIP Periodical Service Platform. The Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) risk‐of‐bias tool was used to evaluate the risk of bias in the included studies. A meta‐analysis was performed based on the guidelines provided in the Cochrane Handbook for Systematic Reviews of Interventions (CHSRI) by using Stata 17.0 software. Nineteen studies were included in the meta‐analysis. Jujube fruit extracts significantly decreased the level of malonaldehyde (MDA) and increased the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px). Meanwhile, there was no significant improvement in the catalase (CAT) levels. In addition, there was considerable heterogeneity in the results of the meta‐analysis. The results of the subgroup analysis indicated that the animal model, type of extracts, and source of target parameters may have contributed to the heterogeneity. Jujube fruit extracts are healthy and effective antioxidant dietary supplements that may be an effective adjunctive therapy for diseases in which oxidative stress is a major pathological factor. However, the overall methodological quality of the included studies was low, and additional research is warranted.

| 5313   ZHU et al.   metabolic syndrome, diabetes, and hyperlipidemia, among others, are considerably influenced by oxidative stress (Le Lay et al., 2014;Masenga et al., 2023;McGill & Hinson, 2020;Morris et al., 2019;Seen, 2021;Unsal et al., 2021).In these diseases, oxidative stress, as a primary cause of pathology or the secondary contributor to disease progression, is a key therapeutic target that requires attention (Forman & Zhang, 2021).Enzymatic and nonenzymatic antioxidant systems, which collectively create an efficient in vivo antioxidant defense system, are the two primary antioxidant systems.Enzymatic antioxidants primarily include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR).These enzymes can effectively resist oxidative damage.Nonenzymatic antioxidants, such as vitamins C and E, can also directly protect against oxidative stress and collaborate with endogenous enzymatic antioxidants.This helps scavenge ROS and boost antioxidant activity more effectively (Jiao et al., 2016).
Oxidative stress, a component of several diseases, has drawn more attention in recent years.However, the development of therapeutic antioxidant approaches has not been very successful.
Researchers are exploring various antioxidant treatment strategies, and supplementation with dietary antioxidants is one of them (Forman & Zhang, 2021).Synthetic antioxidants like butylated hydroxyanisole and butylated hydroxytoluene have been employed in the food sector.However, these antioxidants have been linked to cancer and liver damage (Arunachalam et al., 2022).These developments have led to an increased interest in naturally occurring antioxidants derived from plants.Numerous herbs are considered safe and healthy, and their active components can serve as both enzymatic and nonenzymatic antioxidants.
Jujube (Ziziphus jujuba Mill.), also known as Chinese date, belongs to the genus Ziziphus of the family Rhamnaceae and is native to China.
Of the 170 Ziziphus species, it is the most significant in terms of both ecology and commerce, and it also occupies the greatest cultivated area (Li, Muhammad, et al., 2023;Li, Pan, et al., 2023).Jujube fruit (Figure 1), which has been used in China for more than 4000 years as a food supplement and traditional herbal medicine, is nutritious, has multiple health benefits, and is regarded as an extremely valuable fruit and excellent medicinal herb that can prolong life by promoting enhanced digestion, better sleep, and blood nourishment (Chen & Tsim, 2020).
Among the 8000 odd traditional Chinese medicine (TCM) prescriptions included in the Chinese Medicated Diet Dictionary, jujube fruit appears 400 times, making it the most used TCM herb (Zhu, 2008).
Jujube fruit contains various nutrients, including polysaccharides, polyphenols, amino acids, triterpenic acids, fatty acids, nucleotides, dietary fiber, alkaloids, vitamins, and other nutrients.It has antioxidant, anti-inflammatory, anticancer, antihyperglycemic, antihyperlipidemic, immune regulatory, neuroprotective, sedative, and antiviral properties (Lu et al., 2021).Most of the reported therapeutic effects are primarily attributed to the antioxidant and anti-inflammatory properties of jujube fruit extracts, especially the antioxidant properties.The extracts act as enzymatic and nonenzymatic antioxidants and effectively inhibit ROS production and lipid peroxidation (Hong et al., 2020).Previous studies have shown that jujube fruit extracts may inhibit NF-κB to suppress the expression of inflammatory proteins and activate Nrf2mediated antioxidant responses to ameliorate tissue damage (Huang et al., 2017;Kim et al., 2020).
Jujube fruit extracts have been shown to attenuate oxidative stress in several studies.However, systematic summaries of pertinent data are still lacking.Here, we conducted a systematic review and meta-analysis of rodent experiments, for the first time, to evaluate the effects of jujube extracts on oxidative stress in rodent models, and provide a reference and evidence for the clinical application of jujube extracts as a dietary supplement to improve the outcomes of oxidative stress-related diseases.

| MATERIAL S AND ME THODS
The current systematic review and meta-analysis were designed and carried out following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA).It was registered with PROSPERO (registration number: CRD42023426300).

| Search strategy
The databases of PubMed, Web of Science, Embase, China National Knowledge Infrastructure (CNKI), Wanfang Data Knowledge Service Platform, and VIP Periodical Service Platform were searched from database inception until April 2023.The language was limited to English or Chinese.Medical subject headings (MeSH) and free words for database searches were as follows: [("Ziziphus" OR "Jujube" OR "Chinese jujube" OR "Ziziphus jujuba Mill" OR "jujube polysaccharide") AND ("oxidative stress" OR "antioxidant" OR "superoxide dismutase" OR "glutathione" OR "glutathione peroxidase" OR "malondialdehyde" OR "catalase") AND ("rat" OR "mice" OR "mouse" OR "animal" OR "rodent" OR "murinae")].The specific retrieval strategies are listed in Table S1.

| Inclusion criteria
(1) Model: studies used rodent models; (2) intervention: jujube fruit extracts with all dosage and duration; (3) comparison: the control group was untreated controlled or vehicle controlled; and (4) outcomes: the outcome parameters included at least one of SOD, MDA, GSH-Px, and CAT.

| Data extraction
After importing all the retrieved literature into EndNote X9, duplicates were removed.Two independent researchers conducted pre-
Each entry was identified as "low risk," "high risk," or "unclear risk".
Any disagreement that arose from the evaluation was settled by discussing it with a third researcher.

| Data synthesis and analysis
The STATA software version 17.0 was used to conduct the statistical analysis.Standardized mean difference (SMD) and 95% confidence interval (95% CI) were used to assess continuous outcomes.p < .05 was considered statistically significant.Then, a general classification of effect sizes into small, medium, and large was made using SMD cutoff points of 0.2, 0.5, and 0.8.Random effects models (DerSimonian-Laird) were used to calculate the combined results.
Statistical heterogeneity was assessed using I-squared (I 2 ), with I 2 > 50% indicating significant heterogeneity.Subgroup analysis was performed to assess the sources of interstudy heterogeneity, in which we considered variables including animal model, type of extracts, duration, and source of target parameters.Sensitivity analysis was carried out to assess the overall results' stability.Publication bias was evaluated with the Egger's test if there were at least 10 studies for each outcome.

| Study characteristics
The 19 studies included were published between 2004 and 2021.
However, owing to the differences in the extraction processes and specific technology, the composition of the jujube fruit extracts obtained from the same macerated medium may have differed.The dosage, timing, and duration of intervention with jujube fruit extracts used in the different studies were different.Detailed characteristics of the included studies are listed in Table 1.

| Risk of bias and quality of included studies
The risk of bias scores of all studies ranged from 2 to 4, with one study obtaining two points, nine studies scoring three points, and nine studies scoring four points.Among the 19 studies included, no study described the methods used to generate the allocation sequence, and three studies did not mention the method of randomization (Huang et al., 2017;Resim et al., 2020;Shen et al., 2009).Four studies reported that the baseline characteristics were similar between groups (Feng et al., 2019;Li et al., 2005;Sheng, 2004;Xie et al., 2018).It was not made clear by any of the research whether or not the allocation to the various groups was sufficiently concealed.Seven studies were considered with random housing because the experimental animals were housed in the same environment and put on a free diet (Cai  et al., 2018;Chi et al., 2015;Du & Liu, 2008;Huang et al., 2017;Shen et al., 2009;Wang, 2010;Wang et al., 2012).Adequate information about the blinding of caregivers or investigators was not offered by any of the studies.None of the studies could determine the exact risk of randomization and blinding of outcome evaluation.One study was evaluated to provide incomplete outcome data (Resim et al., 2020), and the primary outcome data of other studies were complete.The results of the two studies were inconsistent with the description provided in the study methods; hence, these studies could have reported selective results, which could be considered high risk (Feng et al., 2019;Xie et al., 2018).No other source deviation was observed in the studies.
The details are provided in Table 2.

| Effects on SOD
The effect of jujube fruit extracts on SOD levels was reported in 24 pairwise comparisons using data from 19 studies.The combined findings indicated that in comparison to that in the control group, the SOD level in the intervention group was considerably higher, and the effect value was large (SMD = 1.07, 95% CI [0.69, 1.44], p < .01).The results of the heterogeneity test indicated significant heterogeneity (I 2 = 78.11%,p < .01)(Figure 3).

| Effects on MDA
The effect of jujube fruit extracts on MDA levels was reported in 22 pairwise comparisons using data from 18 studies.In the intervention group, the level of MDA was considerably lower than that in the control group, and the effect value was large (SMD = −1.54,95% CI [−2.17,-0.91],p < .01).The results of the heterogeneity test indicated significant heterogeneity (I 2 = 89.91%,p < .01)(Figure 4).

| Effects on GSH-Px
The effect of jujube fruit extracts on GSH-Px was reported in 16 pairwise comparisons using data from 13 studies.Compared to that in the control group, the GSH-Px level was significantly higher in the intervention group, and the effect value was large (SMD = 1.28, 95% CI [0.87, 1.69], p < .01).The results of the heterogeneity test indicated significant heterogeneity (I 2 = 75.60%,p < .01)(Figure 5).

| Effects on CAT
The effect of jujube fruit extracts on CAT was reported in 10 pairwise comparisons using data from eight studies.No statistically significant difference was observed between the intervention and control groups concerning the effects on CAT (SMD = 0.68, 95% CI [−0.22,1.58], p = .14).Results of the heterogeneity test indicated significant heterogeneity (I 2 = 89.63%,p < .01)(Figure 6).

TA B L E 1 (Continued)
of SOD, MDA, GSH-Px, and CAT were not statistically significant.
The heterogeneity in the long treatment group of SOD (I 2 = 54.47%,p = .012)and MDA (I 2 = 58.29%,p = .010)was lower, suggesting that the duration of treatment may have partly influenced the heterogeneity.

| Source of target parameters
We divided the source of target parameters into three subgroups: serum, liver, and others (including testes and multiple sources).The results of the subgroup analysis showed that the liver group had the largest effect size on the SOD, MDA, GSH-Px, and CAT levels.
Concerning GSH-Px, the liver group showed better results than the other group, which showed better results than the serum group; the differences were statistically significant (SMD 1.675 vs. SMD

| Sensitivity analysis and publication bias
We examined the sensitivity of SOD, MDA, GSH-Px, and CAT.The results of a leave-one-out sensitivity analysis revealed that the effect sizes were reliable and unaffected by any particular study.
Since the number of included studies was greater than 10, we evaluated the publication bias of SOD, MDA, and GSH-Px (Figure 7).
The Egger publication bias plot showed that the findings for SOD F I G U R E 3 Effects on SOD.

| DISCUSS ION
The purpose of this systematic review and meta-analysis was to assess the effects of jujube fruit extracts on oxidative stress levels in rodent models.Nineteen studies were included.Jujube fruit extracts could significantly decrease MDA levels and increase SOD and GSH-Px levels but exerted no significant effect on CAT levels.In this metaanalysis, the results of these outcome indicators showed a high degree of heterogeneity.According to the results of the subgroup analysis, the animal model was found to be the potential source of heterogeneity for SOD.The type of extracts was identified as the potential source of heterogeneity for MDA.The target parameter source was considered as the potential source of heterogeneity for GSH-Px.
An increasing body of research suggests that oxidative stress plays a major role in the occurrence and progression of multiple diseases and that controlling oxidative stress can significantly enhance biological metabolism and slow down the progression of diseases.Biomarkers are crucial for assessing the progression of oxidative stress and elucidating the pathogenic mechanisms of the disease.Among the antioxidant defense systems, SOD, CAT, and GSH-Px are the best-known biomarkers and major contributors to protection against oxidative damage (Demirci-Çekiç et al., 2022;Lichtenberg et al., 2023;Marrocco et al., 2017).SOD, which catalyzes the conversion of superoxide radicals into oxygen and hydrogen peroxide, is the initial line of defense against ROS damage (Zhao et al., 2021).Hydrogen peroxide is a substrate for CAT and GSH-Px, whereas CAT is the core antioxidant enzyme in most organisms that catalyzes the decomposition of hydrogen peroxide into water and oxygen (Baker et al., 2023), and GSH-Px catalyzes the reduction of hydrogen peroxide or organic hydroperoxides to water or the corresponding alcohol using the appropriate reducing  agent (Pei et al., 2023).These enzymatic antioxidants work collectively to reduce ROS levels and limit their toxicity.Lipids are highly susceptible to oxidative stress, and MDA, as a comparatively stable metabolite during lipid peroxidation between ROS and polyunsaturated fatty acids, is considered to be a typical biomarker of lipid peroxidation.Changes in MDA can reflect the degree of oxidative stress; hence, this molecule is commonly used as a measure of oxidative stress (Li, Muhammad, et al., 2023;Li, Pan, et al., 2023;Mas-Bargues et al., 2021).Jujube fruit extracts can reduce the levels of MDA and increase the levels of SOD and GSH-Px, indirectly confirming that they can reduce the levels of lipid peroxidation and activate the inherent antioxidative enzyme system to balance the oxidative stress status in multiple tissues and organs.
However, jujube fruit extracts did not significantly raise CAT levels in the included studies.Possibly, the heterogeneity of the included literature and its limited scope prevented jujube fruit extracts from playing a larger role in this regard.It is worth mentioning that jujube is rich in nonenzymatic plant compounds such as vitamins (especially vitamin C), polyphenols, and triterpenoids (Lu et al., 2021), and while these nonenzymatic antioxidants may not be able to outcompete enzymatic antioxidants that can catalyze the depletion of ROS, they are equally indispensable (Meulmeester et al., 2022).
The findings of our subgroup analysis suggested that the animal model may influence the antioxidant effect.When the researchers used toxicity models, jujube fruit extracts exhibited a better effect on oxidative stress levels, perhaps because the mechanism of action of oxidative stress is different in different diseases.In toxic diseases, oxidative stress frequently plays a significant role because toxins trigger excessive ROS production, which results in oxidative damage (McGill & Hinson, 2020;Unsal et al., 2021).In addition to this, oxidative stress is merely one of the many factors that influence multiple diseases, and it develops after other causes have already triggered the pathology.
Oxidative stress interferes with many signaling pathways, thereby affecting various biological processes and aggravating the symptoms of diseases by modifying proteins, fostering inflammation, triggering apoptosis, and deregulating autophagy, among other mechanisms (Forman & Zhang, 2021).It is worth mentioning that exercise is one of the common physiological conditions linked to increased oxidative stress, and high-intensity exercise leads to an overproduction of ROS and oxidative damage to muscle fibers (Arazi et al., 2021).
The type of jujube extract also affects the antioxidant effect.
People extract the desired natural active ingredients from jujube through a series of processes, in which jujube polysaccharide is the primary research object (Wang, 2021).Notably, both crude extracts The antioxidant effect of jujube extracts could be exerted on blood and different tissues and organs, and its effect on the liver is most significant.All data support this result.The human body is a unified whole, with various tissues and organs interacting with each other and each having its unique properties.A crucial metabolic organ, the liver has an abnormal buildup of lipids, metabolic products, and toxins that can disrupt the redox state and harm cells (Sadasivam et al., 2022).
This implies that the liver is susceptible to oxidative stress and is also an important target for drug action.Blood travels through the body, exchanging substances with various organs and tissues.The oxidative stress index in serum can reflect the overall oxidative stress state to a certain extent.The outcomes of the subgroup analyses suggested that jujube fruit extracts were effective in regulating systemic oxidative stress.Although these extracts demonstrated particularly strong efficacy in the liver, this notable effect may be attributed to the inherent characteristics of the liver itself.Researchers have primarily focused on the liver to determine oxidative stress levels, and there is insufficient data on other tissues and organs.This knowledge gap is worthy of further exploration.
In the included studies, the timing of the therapeutic intervention is varied.However, regardless of whether the jujube fruit extracts were used as a preventive drug or directly to treat disease, the results indicated the good antioxidant capacity of jujube fruit extracts.
In many of the included studies, the dosage of jujube extracts was categorized into different levels.The results of most of these studies indicated that the dose effect of jujube extracts showed a curve in which the antioxidant effect of medium doses was superior to that of high doses, which in turn was superior to that of low doses.It tentatively demonstrated a dose-effect relationship between jujube fruit extracts and their antioxidant capacity.Owing to the substantial differences in dose distribution in the included studies, we combined all doses in the study, which, to some extent, weakened the therapeutic effect of jujube fruit extracts.
Multiple dietary supplements are touted as antioxidants, but only a few have been shown to promote health benefits.Similarly, multiple plant extracts exhibit antioxidant activity, but not all of them can be used as dietary supplements (Ali et al., 2020).Quality, safety, and efficacy are important factors of consideration for dietary supplements (Dwyer et al., 2018;Féart, 2020).
Keeping quality control-related factors aside, the efficacy of the jujube fruit has been demonstrated in clinical applications, where it is often used to treat endocrine, cardiovascular, psychiatric, gastrointestinal, and respiratory disorders (Wang, 2021).However, as with most botanical dietary supplements, the type and amount of evidence for the efficacy of jujube fruit is limited, and the vast majority of evidence now focuses on the antioxidant effects of jujube fruit.We combined different doses of jujube fruit extracts and performed a meta-analysis while underestimating the antioxidant effect.The results indicated that the extracts could still significantly modulate the levels of SOD, MDA, and GSH-Px, and the effect values were all large, which confirms the effectiveness of the jujube fruit extracts in resisting oxidative stress.Jujube is one of the most commonly used herbs in TCM treatments as well as a fruit that can be consumed daily, and its extensive use over centuries indicates its safety.No study has reported the side effects of jujube.Jujube is susceptible to infestations by fungi such as Aspergillus niger during growth and ripening.However, even though the quality of the jujube fruit can be affected by A. niger, owing to the characteristics of the fruit, it does not accumulate ochratoxin A, a toxic secondary fungal metabolite that is widespread in food and agricultural products (Xin et al., 2023).Based on these characteristics, we can determine that jujube is an excellent antioxidant dietary supplement.In addition, the jujube fruit has a high yield, good flavor, and reasonable price and is easy to store.Of the many antioxidant dietary supplements available, it may not be the most potent in terms of its antioxidant capacity, but it is certainly one of the healthiest and most easily accessible fruits available to the general public.
This study had several limitations.First, the number of studies in which the effects of jujube fruit extracts on oxidative stress were evaluated was small.Second, the majority of the included studies had poor quality, and there were substantial interstudy differences and high heterogeneity of results.Finally, a publication bias for MDA and GSH-Px was observed, which might have caused the benefits of jujube fruit extracts to be overestimated.

| CON CLUS ION
Jujube fruit extracts are healthy and effective antioxidant dietary supplements with beneficial effects on SOD, GSH-Px, and MDA levels.These extracts may be an effective adjunctive therapy for diseases in which oxidative stress is an important pathological factor.
However, the overall methodological quality of the included studies liminary screening based on title and abstract to exclude irrelevant literature, and the rest were reviewed by obtaining the complete text to further weed out literature that did not match the inclusion criteria.Disputes regarding the inclusion of a study were settled by discussing with a third researcher.Details were extracted from selected studies: (1) the first author and publication year; (2) characteristics of experimental animals, including animal species, sex, sample size, and weight; (3) the models and modeling methods; (4) information regarding intervention and control groups (administration, dosage, and duration of intervention); and (5) outcome indicator and sources of target parameter.For studies requiring data extraction from images, we used GetData Graph Digitizer software (version 2.26) to extract the corresponding outcome indicators.To categorize therapeutic drugs into subgroups based on data from each original study, we used the Cochrane Handbook for Systematic Reviews of Interventions (CHSRI) and grouped the findings from various subgroups into a single treatment group for analysis.When extracting data of the identical outcome indicator obtained from multiple test samples, we used the mean value.
1.122 vs. SMD 0.571, p = .025).The heterogeneity of SOD in the serum group was low (I 2 = 52.11%,p = .037).The heterogeneity of SOD (I 2 = 32.57%,p = .157)and GSH-Px (I 2 = 26.68%,p = .207)in the liver group was significantly lower, suggesting that the source of the target parameters could partially explain the heterogeneity of the study data.

F
Effects on GSH-Px.F I G U R E 6 Effects on CAT.TA B L E 3 Subgroup analyses in SOD, MDA, GSH-Px, and CAT with the ex ante parameters.
Flowchart for selection of studies.Basic characteristics of included studies.
TA B L E 1

CI] p for meta-analysis I 2 (%) p for heterogeneity
jujube obtained by simple extraction and polysaccharides, triterpene acid, and flavonoids obtained by complex isolation and purification processes have good antioxidant effects.At present, the primary medium used for extracting active ingredients from jujube is water or ethanol.Owing to the different polarities and solubilities of the target bioactive ingredients, appropriate extraction methods must be used.
F I G U R E 7 Egger's publication bias plot for SOD, MDA, and GSH-Px. of tracts.However, the effect of ethanol extracts on MDA, GSH-Px, and CAT was considerably better than that of water extracts, which may indicate that the substances soluble in ethanol in jujube can better regulate the levels of oxidative stress.However, these findings need further validation from more relevant studies.